Moist Heat Disinfection of HBV

Krugman and co-workersKobayashi and co-workersLelie and co-workers
Year of Publication197919841987
Initial serum of HBV
    StrainMS-2JHB001NYBC 78-564
    Infectivity (units/mL)107.5108107
    Heat-inactivated sampleInoculum IIInoculum III
    Dilution of initial serum1:10 using sterile distilled water1:1000 using phosphate bufferInoculum I*** was spiked using HBV serum (NYBC 78-564) so that the final infectivity titer would be 105 CID50/mL. This HBV-spiked solution underwent moist heat disinfection at 103 °C for 90 s. This heat-inactivated solution was ultracentrifuged and was sterile-filtered to produce inoculum II.Inoculum I*** underwent moist heat disinfection at 103 °C for 90 s under production circumstances and was diluted in PBS so that the protein concentration would be 1 mg/mL. This protein solution was spiked using HBV serum (NYBC 78-564) so that the final infectivity titer would be 105 CID50/mL. This HBV-spiked protein solution underwent moist heat disinfection at 65 °C for 10 h to produce inoculum III.
    Volume20 mL1 mL50 mL50 mL
    Infectivity titer (CID50/mL)106.5105105105
Heat-inactivation method
    HeatingElectric burnerLiquid paraffin bath at 98 °COil bathWater bath at 65 °C
    CoolingNAIce water bathIce bathNA
    ThermometryThermometerThermocoupleData-logger apparatus (Kaye Instruments)Data-logger apparatus
    Come-up phase43 s4 min4340 as A0 valueNA
    Holding time phase98 °C for 1 min (A0 value: 3786)98 °C for 2 min (A0 value: 7571)19762 as A0 value (Temperature range: 103.0 to 104.1 °C)65 °C for 10 hr (A0 value: 1138)
    Come-down phase25 min to the room tempCooled immediately763 as A0 valueNA
    Estimated A0 valueConservatively estimated as 3786Conservatively estimated as 7571248651138
    Inoculated volume0.1 mL1 mL10 mL10 mL
    Inoculated subjects29 susceptible human volunteersTwo susceptible chimpanzeesTwo susceptible chimpanzees
    Comments With informed consents conformed to the World Medical Association's Draft Code of Ethics on Human Experimentation (1961)A male weighing 10.3 kg and a female weighing 8.8 kg from West Africa. Both were negative for HBsAg, anti-HBs and anti-HBc (RIA).Two wild-caught chimpanzees aged 3–5 yr and negative for HBsAg, anti-HBs and anti-HBc (RIA). The mixture of inoculum II and III was administered to each of the two susceptible chimpanzees.
    Follow-up period8 and a half months30 weeks28 weeks
    Liver blood chemistryAll normalAll normalAll normal
    HBsAg (CF or RPHA)All negative*NANA
    HBsAg (RIA)26 negatives & 3 positives**All negativeAll negative
    anti-HBs (RIA)NAAll negativeAll negative
    anti-HBc (RIA)NAAll negativeAll negative
    Liver biopsyNAAll normalAll normal
    Comments*Reported in 1971 and 1973, using the test methods available at the time. **Report in 1979. The stored samples were retested using more sensitive radioimmunoassay method (Ausria II).Liver blood chemistry tests: total protein, bilirubin, alkaline phosphatase, asparate and alanine aminotransferases, lactic dehydrogenase, γ-glutamine transpeptidase, cholinesterase, leucine aminopeptidase, and triglycerides. HBV markers were tested by radioiimunoassay (Ausria II, Ausab, and Corab, respectively; Abbott Laboratories, North Chicago, IL.)***HBV infectivity was eliminated using chemical purification step followed by two steps of moist heat disinfection at 103 °C for 90 s and at 65 °C for 10 h, respectively. Inoculum I, II, and III were prepared under the same circumstances to produce the vaccine. HBV serum (NYBC 78-564) was diluted using 20 L of plasma negative for HBV markers as the starting material. Inoculum I was the ultracentrifuge resuspension in PBS. Elimination of HBV infectivity in inoculum I was confirmed using two chimpanzees.Liver blood chemistry tests: ALT (alanine aminotransferases) and AST (asparate aminotransferases) detected by kinetic spectrophotometric assay with an Abbott ABA-100 Analyzer using Abbott reagents and reference standards. HBV markers were tested by radioimmunoassay (Ausria II, Ausab, Corab, Abbott Labs).
  • NA: not available, CF: complement fixation, RPHA: reverse passive hemaglutination, RIA: radioimmunoassay, HBsAg: hepatitis B antigen, anti-HBs: hepatitis B antibody, anti-HBc: antibody to hepatitisB core antigen. (References 15–22).